Picric acid (2,4,6-trinitrophenol) is a compound used in a variety of industrial applications including the manufacture of explosives, aniline, color fast dyes, pharmaceuticals and in steel etching. Picric acid and ammonium picrate were first obtained as fast dyes for silk and wool. However, the unstable nature of picric acid was soon exploited for use as an explosive and explosive boosters where it is the primary component of blasting caps which are used for the detonation of 2,4,6-Trinitrotoluene (TNT). Because of its explosive nature, disposal of waste picric acid poses unique hazards not generally associated with other environmental toxicants.
Mounting public concern and increasing government regulations have provided the impetus for a safe, effective means to remediate picric acid contaminated environments. Past methods of disposing of munitions and other wastes containing picric acid have included dumping at specified land-fill areas, isolation in suitable, reinforced containers, land based deep-welling, dumping in deep water at sea and incineration. All of these methods carry some potential for harm to the environment. For example, incineration creates a problem of air pollution and disposal on land risks the possibility that toxic substances will elute or leach into locations where they may threaten aquatic life forms, animals or humans. A more desirable disposal method might incorporate a chemical or enzymatic degradative process.
The metabolic reduction of organic nitrogen groups has been known for some time. Westfall, J. Pharmacol. Exp. Therap., 78:386 (1943) reported that liver, kidney and heart tissue are active in the reduction of trinitrotoluene, however, was not able to identify the specific enzyme system responsible. Westerfield et al., J. Biol. Chem., 227:379 (1957) further disclosed that purified xanthine oxidase is capable of reducing organic nitrogen groups and demonstrated that the molybdenum (Mo) co-factor was essential in the degradative process.
Microbial degradation of organic nitrogen compounds has been limited to a handful of organisms. Erickson, J. Bact., 41:277 (1941) reported that certain strains of Micromonospora were able to utilize picric acid and trinitro-resorcinol as a carbon source and Moore. J. Gen. Microbiol., 3:143 (1949) described two unspecified proactinomnycetes as being capable of using nitrobenzene as a simultaneous source of carbon and nitrogen. Gundersden et al., Acta. Agric. Scand., 6:100 (1956) described the metabolism of picric acid by Corynebacterium simplex which was isolated from soil as a 4,6-dinitro-2-methylphenol-degrading organism. Degradation was determined by measuring the amount of nitrate produced when the organism was contacted with an organic nitrogen compound. The extent of degradation and the identification of specific degradation products were not reported. Later, Wyman et al., Appl. Environ. Microbiol., 37(2):222 (1979) found that a strain of pseudomonas aeruginosa reduced picric acid to 2-amino-4,6-dinitrophenol (picramic acid) under anaerobic conditions. Wyman further determined that degradation products from both picric and picramic acid produced by this strain demonstrated mutagenicity as assayed by the standard AMES test. Another pseudomonas sp., P. putida, has been shown to be able to use picric acid as a carbon source and achieve some bio-conversion of the compound to 1,3,5-trinitro benzene, 2,4,6-trinitroaldehyde, and 3,5-dinitrophenol. Kearney et al., Chemosphere, 12 (11-12):1583 (1983).
Most recently, Rhodococcus erythropolis has been identified as a picric acid degrading bacteria. Lenke et al., Appl. Environ. Microbiol., 58(9):2933 (1992) teach that R. erythropolis, under aerobic conditions, can incompletely utilize picric acid as a nitrogen source producing nitrite and 2,4,6-trinitro-cyclohexanone, which cannot be degraded further.
In spite of the investigative activity in the area of microbial degradation of picric acid and other organic nitrogen compounds, there remain several difficulties to overcome before any of the above mentioned microbial systems can be used for the effective remediation of contaminated environments. All of the microbes investigated are isolated organisms and, although they show picric acid degrading activity in vitro, there is little evidence that these organisms will function under in situ conditions. Additionally, no organism or group of organisms has been isolated that demonstrate complete degradation of picric acid. At present the art teaches that only partial degradation is possible and that some of the degradation products may also be harmful to the environment as mutagens. There remains a need, therefore, for an effective degradative process for picric acid and related compounds that will degrade those compounds completely and be effective in both the in vitro and in situ remediation of contaminated environments.